Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development

Abstract

Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development.

Key message Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.

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Abbreviations

GUS:

Beta-d-glucuronidase gene

References

  1. Alba R, Payton P, Fei Z, McQuinn R, Debbie P, Martin GB, Tanksley SD, Giovannoni JJ (2005) Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. Plant Cell 17:2954–2965. doi:10.1105/tpc.105.036053

    PubMed  Article  CAS  Google Scholar 

  2. Aoki K, Yano K, Suzuki A, Kawamura S, Sakurai N, Suda K, Kurabayashi A, Suzuki T, Tsugane T, Watanabe M, Ooga K, Torii M, Narita T, Shin IT, Kohara Y, Yamamoto N, Takahashi H, Watanabe Y, Egusa M, Kodama M, Ichinose Y, Kikuchi M, Fukushima S, Okabe A, Arie T, Sato Y, Yazawa K, Satoh S, Omura T, Ezura H, Shibata D (2010) Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics. BMC Genomics 11:210. doi:10.1186/1471-2164-11-210

    PubMed  Article  Google Scholar 

  3. Beaudoin N, Rothstein SJ (1997) Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato. Plant Mol Biol 33:835–846. doi:10.1023/A:1005773722657

    PubMed  Article  CAS  Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3

    PubMed  Article  CAS  Google Scholar 

  5. Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308. doi:10.1038/nbt.1506

    PubMed  Article  CAS  Google Scholar 

  6. Chen Y, Wang A, Zhao L, Shen G, Cui L, Tang K (2009) Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits. Biotechnol Appl Biochem 52:303–312. doi:10.1042/BA20080054

    PubMed  Article  CAS  Google Scholar 

  7. Cordes S, Deikman J, Margossian LJ, Fischer RL (1989) Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato. Plant Cell 1:1025–1034. doi:10.1105/tpc.1.10.1025

    PubMed  CAS  Google Scholar 

  8. Coupe SA, Deikman J (1997) Characterization of a DNA-binding protein that interacts with 5′ flanking regions of two fruit-ripening genes. Plant J 11:1207–1218. doi:10.1046/j.1365-313X.1997.11061207.x

    PubMed  Article  CAS  Google Scholar 

  9. Deikman J, Fischer RL (1988) Interaction of a DNA binding factor with the 5′-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO J 7:3315–3320

    PubMed  CAS  Google Scholar 

  10. Deikman J, Kline R, Fischer RL (1992) Organization of ripening and ethylene regulatory regions in a fruit-specific promoter from tomato (Lycopersicon esculentum). Plant Physiol 100:2013–2017. doi:10.1104/pp.00.4.2013

    PubMed  Article  CAS  Google Scholar 

  11. Deikman J, Xu R, Kneissl ML, Ciardi JA, Kim KN, Pelah D (1998) Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5′-flanking region of the ripening-related E8 gene. Plant Mol Biol 37:1001–1011. doi:10.1023/A:1006091928367

    PubMed  Article  CAS  Google Scholar 

  12. Dharmapuri S, Rosati C, Pallara P, Aquilani R, Bouvier F, Camara B, Giuliano G (2002) Metabolic engineering of xanthophyll content in tomato fruits. FEBS Lett 22:30–34. doi:10.1016/S0014-5793(02)02699-6

    Article  Google Scholar 

  13. Estornell LH, Orzáez D, López-Peña L, Pineda B, Antón MT, Moreno V, Granell A (2009) A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits. Plant Biotechnol J 7:298–309. doi:10.1111/j.1467-7652.2009.00402.x

    PubMed  Article  CAS  Google Scholar 

  14. Ferrie BJ, Beaudoin N, Burkhart W, Bowsher CG, Rothstein SJ (1994) The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening. Plant Physiol 106:109–118. doi:10.1104/pp.106.1.109

    PubMed  Article  CAS  Google Scholar 

  15. Gaffe J, Tiznado ME, Handa AK (1997) Characterization and functional expression of a ubiquitously expressed tomato pectin methylesterase. Plant Physiol 114:1547–1556. doi:10.1104/pp.114.4.1547

    Google Scholar 

  16. Hall LN, Bird CR, Picton S, Tucker GA, Seymour GB, Grierson D (1994) Molecular characterisation of cDNA clones representing pectinesterase isozymes from tomato. Plant Mol Biol 25:313–318. doi:10.1007/BF00039542

    PubMed  Article  CAS  Google Scholar 

  17. Hirai T, Fukukawa G, Kakuta H, Fukuda N, Ezura H (2010) Production of recombinant miraculin using transgenic tomato in a closed-cultivation system. J Agric Food Chem 58:6096–6101. doi:10.1021/jf100414v

    PubMed  Article  CAS  Google Scholar 

  18. Hiwasa-Tanase K, Hirai T, Kato K, Duhita N, Ezura H (2012) From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants. Plant Cell Rep 31:513–525. doi:10.1007/s00299-011-1197-5

    PubMed  Article  CAS  Google Scholar 

  19. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  20. Kausch KD, Handa AK (1995) Molecular cloning and nucleotide sequence of a lipoxygenase cDNA from ripening tomato fruit. Plant Physiol 107:669–670. doi:10.1104/pp.107.2.669

    PubMed  Article  CAS  Google Scholar 

  21. Kneissl ML, Deikman J (1996) The tomato E8 gene influences ethylene biosynthesis in fruit but not in flowers. Plant Physiol 112:537–547. doi:10.1104/pp.112.2.537

    PubMed  CAS  Google Scholar 

  22. Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990) An improved assay for β-glucuronidase (GUS) in transformed cells: methanol almost suppresses a putative endogenous GUS activity. Plant Sci 70:133–140. doi:10.1016/0168-9452(90)90042-M

    Article  CAS  Google Scholar 

  23. Le LQ, Lorenz Y, Scheurer S, Fötisch K, Enrique E, Bartra J, Biemelt S, Vieths S, Sonnewald U (2006) Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. Plant Biotechnol J 4:231–242. doi:10.1111/j.1467-7652.2005.00175.x

    PubMed  Article  CAS  Google Scholar 

  24. Lewinsohn E, Schalechet F, Wilkinson J, Matsui K, Tadmor Y, Nam KH, Amar O, Lastochkin E, Larkov O, Ravid U, Hiatt W, Gepstein S, Pichersky E (2001) Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol 127:1256–1265. doi:10.1104/pp.010293

    PubMed  Article  CAS  Google Scholar 

  25. Lincoln JE, Cordes S, Read E, Fischer RL (1987) Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fruit development. Proc Natl Acad Sci USA 84:2793–2797

    PubMed  Article  CAS  Google Scholar 

  26. Matsukura C, Aoki K, Fukuda N, Mizoguchi T, Asamizu E, Saito T, Shibata D, Ezura H (2008) Comprehensive resources for tomato functional genomics based on the miniature model tomato Micro-Tom. Curr Genomics 9:436–443. doi:10.2174/138920208786241225

    PubMed  Article  CAS  Google Scholar 

  27. Mollet B, Niederberger P, Pétiard V (2008) Novel tomato flavours introduced by plastidial terpenoid pathway engineering. Trends Biotechnol 26:4–6. doi:10.1016/j.tibtech.2007.10.004

    PubMed  Article  CAS  Google Scholar 

  28. Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL (1993a) Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proc Natl Acad Sci USA 90:5939–5943

    PubMed  Article  CAS  Google Scholar 

  29. Montgomery J, Pollard V, Deikman J, Fischer RL (1993b) Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp. Plant Cell 5:1049–1062. doi:10.1105/tpc.5.9.1049

    PubMed  CAS  Google Scholar 

  30. Moon H, Callahan AM (2004) Developmental regulation of peach ACC oxidase promoter–GUS fusions in transgenic tomato fruits. J Exp Bot 55:1519–1528. doi:10.1093/jxb/erh162

    PubMed  Article  CAS  Google Scholar 

  31. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 10:4321–4325. doi:10.1093/nar/8.19.4321

    Article  Google Scholar 

  32. Nicholass FJ, Smith CJ, Schuch W, Bird CR, Grierson D (1995) High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol Biol 28:423–435. doi:10.1007/BF00020391

    PubMed  Article  CAS  Google Scholar 

  33. Orzaez D, Mirabel S, Wieland WH, Granell A (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol 140:3–11. doi:10.1104/pp.105.068221

    PubMed  Article  CAS  Google Scholar 

  34. Ozaki S, Ogata Y, Suda K, Kurabayashi A, Suzuki T, Yamamoto N, Iijima Y, Tsugane T, Fujii T, Konishi C, Inai S, Bunsupa S, Yamazaki M, Shibata D, Aoki K (2010) Coexpression analysis of tomato genes and experimental verification of coordinated expression of genes found in a functionally enriched coexpression module. DNA Res 17:105–116. doi:10.1093/dnares/dsq002

    PubMed  Article  CAS  Google Scholar 

  35. Pear JR, Sanders RA, Summerfelt KR, Martineau B, Hiatt WR (1993) Simultaneous inhibition of two tomato fruit cell wall hydrolases, pectinmethylesterase and polygalacturonase, with antisense gene constructs. Antisense Res Dev 3:181–190

    PubMed  CAS  Google Scholar 

  36. Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R, Bouvier F, Camara B, Giuliano G (2000) Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J 24:413–419. doi:10.1104/pp.105.068221

    PubMed  Article  CAS  Google Scholar 

  37. Schijlen E, Ric de Vos CH, Jonker H, van den Broeck H, Molthoff J, van Tunen A, Martens S, Bovy A (2006) Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit. Plant Biotechnol J 4:433–444. doi:10.1111/j.1467-7652.2006.00192.x

    PubMed  Article  CAS  Google Scholar 

  38. Schijlen EG, de Vos CH, Martens S, Jonker HH, Rosin FM, Molthoff JW, Tikunov YM, Angenent GC, van Tunen AJ, Bovy AG (2007) RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits. Plant Physiol 144:1520–1530. doi:10.1104/pp.107.100305

    PubMed  Article  CAS  Google Scholar 

  39. Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. doi:10.1093/pcp/pci251

    PubMed  Article  CAS  Google Scholar 

  40. Sun HJ, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotechnol J 5:768–777. doi:10.1111/j.1467-7652.2007.00283.x

    PubMed  Article  CAS  Google Scholar 

  41. Wang S, Liu J, Feng Y, Niu X, Giovannoni J, Liu Y (2008) Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. Plant J 55:89–103. doi:10.1111/j.1365-313X.2008.03489.x

    PubMed  Article  CAS  Google Scholar 

  42. Xu R, Goldman S, Coupe S, Deikman J (1996) Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements. Plant Mol Biol 31:1117–1127. doi:10.1007/BF00040829

    PubMed  Article  CAS  Google Scholar 

  43. Yano K, Watanabe M, Yamamoto N, Tsugane T, Aoki K, Sakurai N, Shibata D (2006) MiBASE: a database of a miniature tomato cultivar Micro-Tom. Plant Biotechnol. 23:195–198. doi:10.5511/plantbiotechnology.23.195

    Article  CAS  Google Scholar 

  44. Yano M, Hirai T, Kato K, Hiwasa-Tanase K, Fukuda N, Ezura H (2010) Tomato is a suitable material for producing recombinant miraculin protein in genetically stable manner. Plant Sci 178:469–473. doi:10.1016/j.plantsci.2010.02.016

    Article  CAS  Google Scholar 

  45. Zhang H, Zhao L, Chen Y, Cui L, Ren W, Tang K (2007) Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum). Biotechnol Appl Biochem 48:101–107. doi:10.1042/BA20060224

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

We thank the members of the Ezura laboratory for helpful discussions. Micro-Tom seeds (TOMJPF00001) were obtained from the National BioResource Project Tomato (NBRP-Tomato). This research was supported through grants from the “Development of Fundamental Technologies for the Production of High-Value Materials Using Transgenic Plants” project of the Ministry of Economy, Trade, and Industry of Japan to H.E. and K.T.

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Correspondence to Hiroshi Ezura.

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Communicated by H. Ebinuma.

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Hiwasa-Tanase, K., Kuroda, H., Hirai, T. et al. Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development. Plant Cell Rep 31, 1415–1424 (2012). https://doi.org/10.1007/s00299-012-1257-5

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Keywords

  • Fruit-specific promoter
  • Tomato
  • Green stage
  • Red stage
  • Fruit development